Apparatus and Method for Magnetically Separating Cells From Mixture

ABSTRACT

According to the conventional process of separating necessary cells from a cell mixture containing specific cells tagged with magnetic beads, there is a problem in that separation process is complicated when the separation efficiency is high whereas the separation efficiency is relatively low when the separation process is simplified. The present invention provides an apparatus and method for separating cells from a cell mixture simply and efficiently through the processes of creating a cell mixture layer between upper and lower plates by adjusting a gap between the upper and lower plates and of adjusting a thickness of the created layer and separating the layer while applying a magnetic field to the layer from the upper plate, wherein the lower plate is provided with a cell mixture holding portion, in which the cell mixture containing specific cells tagged with magnetic beads is accommodated in an upwardly convex shape, at a top surface thereof and the upper plate is positioned above the lower plate to face each other and to adsorb the cell mixture accommodated in the cell mixture holding portion of the lower plate into a bottom surface thereof.

TECHNICAL FIELD

The present invention relates to an apparatus and method for separatingcells from a cell mixture, and more particularly, to an apparatus andmethod for separating necessary cells from a cell mixture by applying amagnetic field to the cell mixture in which specific cells tagged withmagnetic carriers are mixed.

BACKGROUND ART

In U.S. Pat. No. 6,602,422 (entitled “micro column system”), if amagnetic field is applied to a column from the outside while a cellmixture in which specific cells tagged with magnetic beads (serving as amagnetic carrier) are mixed is poured into the column with small steelballs filled therein in a gravitational direction, the specific cellsare adsorbed while flowing through gaps between the magnetized steelballs but the other cells flow downward. At this time, the specificcells which have been bound to the steel balls and thus separated fromthe mixture are recovered by eliminating the applied magnetic field andthen causing a buffer solution to flow.

However, when a user intends to separate the cells with a variety ofsizes using the column type cell separation apparatus, the used steelballs should be changed in accordance with the sizes of magnetic beadsor specific cells and the adsorption intensity for the specific cells tobe separated since the movement of cells may be hindered by the size ofgaps between the steel balls.

Further, since the cells mixed in the cell mixture gather around thegaps caused by the accumulated steel balls, it may be difficult torecover the specific cells separated from the mixture.

Furthermore, in order to prevent the gaps between the steel balls frombeing choked as the cells coagulate, the steel balls in the cellseparation apparatus should be periodically shaken to be homogeneoususing a pipette during the separation process.

In U.S. Pat. No. 5,602,042 (entitled “method and apparatus formagnetically separating biological particles from a mixture”),biological particles tagged with beads are separated from a mixture byadhering to a plate by means of an applied magnetic field while aseparation means including a magnet and a plate is vertically moved androtated in a state where the separation means is immersed in the mixturewith which a closed container.

However, in a case where biological particles are separated using thecontainer type cell separation apparatus, other biological particlesadhering to an outer side of a housing of the separation means arecollected onto the plate while taking the separation means out of themixture. Thus, its separation efficiency will be greatly lowered.

In addition, since the biological particles are separated in a statewhere the mixture is filled in the closed container, a large amount ofsamples are needed to thereby incur high costs and the separation meansfor implementing a vertical and rotational moving mechanism iscomplicated. Further, since an exposed portion such as a housing of theseparation means is brought into direct contact with the mixture, thepossibility of contamination of the mixture will be increased.

In Korean Patent Application No. 2004-25421 (entitled “apparatus andmethod for isolating cells using droplet type cell suspension”),therefore, a cell mixture containing specific cells tagged with magneticbeads is formed into a droplet type cell mixture and a magnetic field isthen applied to the droplet type cell mixture such that the cell mixtureis divided into the specific cells positioned at an upper portionthereof and the other cells positioned at a lower portion thereof. Then,only a buffer solution is additionally supplied to the droplet type cellmixture to completely isolate the lower other cells by means of gravityand then to recover the isolated specific cells. Accordingly, theconfiguration and process of the cell isolation apparatus can besimplified.

However, in a case where the specific cells are isolated using thedroplet type cell mixture, the configuration and process of an apparatusfor forming the cell mixture into a droplet type cell mixture arecomplicated.

Further, since the cell mixture is suspended and thus exposed to theair, the lower cells other than the specific cells may fluctuate.Furthermore, when the buffer solution is additionally added to removethe lower cells other than the specific cells to be isolated, a portionof the lower other cells may be again mixed with the upper specificcells. Thus, there is a problem in that isolation efficiency is lowered.

On the other hand, in a case where the other cells are bound to asurface where the specific cells are recovered, it may have a badinfluence on the subsequent test in which the recovered specific cellswill be used. Therefore, the purity of the obtained specific cells needsto be enhanced to the utmost.

DISCLOSURE OF INVENTION Technical Problem

An object of the present invention is to provide an apparatus and methodfor separating cells in a simple and highly efficient manner through theprocess of creating a cell mixture layer by adjusting a gap between anupper plate and a lower plate oppositely positioned below the upperplate to contain a cell mixture between the upper and lower plates andadjusting a width of the formed cell mixture layer or separating thecell mixture layer while applying a magnetic field to the upper plateover the cell mixture layer.

Technical Solution

According to an aspect of the present invention for achieving theobject, there is provided a cell separation apparatus, comprising alower plate provided with a cell mixture holding portion, in which acell mixture containing specific cells tagged with magnetic carriers isaccommodated in an upwardly convex shape, at a top surface thereof; anupper plate positioned above the lower plate to face each other and toadsorb the cell mixture accommodated in the cell mixture holding portionof the lower plate into a bottom surface thereof; a magnetic fieldapplying means positioned on a top surface of the upper plate; and a gapadjusting means coupled to the upper or lower plate to adjust a gapbetween the upper and lower plates to be increased or decreased, whereinthe gap between the upper and lower plates is decreased by the gapadjusting means such that the cell mixture accommodated in the cellmixture holding portion is adsorbed in the bottom surface of the upperplate and then formed into a cell mixture layer, and the gap between theupper and lower plates is increased by the gap adjusting means such thatthe specific cells moved toward the upper plate by means of a magneticfield applied to the created cell mixture layer through the magneticfield applying means and cells other than the specific cells movedtoward the lower plate by means of gravity are divided and thenpositioned in the bottom surface of the upper plate and the cell mixtureholding portion of the lower plate, respectively.

Preferably, a cell mixture adsorbing portion is formed at the bottomsurface of the upper plate such that the cell mixture holding portion ofthe lower plate is positioned to correspond to the cell mixtureadsorbing portion of the upper plate.

The cell separation apparatus of the present invention may furthercomprise an upper housing with an open bottom and a lower housing withan open top, wherein the magnetic field applying means and the upperplate are installed within the upper housing such that the magneticfield applying means is positioned on the top surface of the upperplate, the lower plate and the gap adjusting means are installed withinthe lower housing such that the gap adjusting means is coupled to thelower plate, and the lower plate is vertically moved by the gapadjusting means to adjust the gap between the upper and lower plates ina state where the upper housing is coupled with the lower housing suchthat the bottom surface of the upper plate and the cell mixture holdingportion of the lower plate are positioned to face each other.

Preferably, the gap adjusting means comprises a lower plate supportformed with a recess for accommodating the lower plate therein at a topside thereof and a bolt-sh aped connection at a bottom side thereof, anda lower plate support moving dial having a nut-shaped connectionthreadedly engaged with the bolt-shaped connection of the lower platesupport; and the lower plate support is vertically moved by turning orrotating the lower plate support moving dial.

Preferably, the gap adjusting means further comprises a dial stopper forrestricting the rotation of the lower plate support moving means suchthat the cell mixture layer can be maintained.

Preferably, the dial stoppers are installed on a bottom surface of thelower plate support moving dial and a predetermined portion of the lowerhousing such that the dial stopper is brought into contact with thebottom surface of the lower plate support moving dial at a positionwhere the rotation of the lower plate support moving dial should beprevented.

Preferably, the gap adjusting means comprises a lower plate supportformed with a recess for accommodating the lower plate therein at a topside thereof and a roller at a bottom side thereof, a lower platesupport moving bar for vertically moving the lower plate support in sucha manner that the roller of the lower plate support is brought intocontact with a plurality of steps with different levels decreasing fromone side to another side, and bar moving dial having a pinion portionmeshed with a rack portion formed on a side surface of the lower platesupport moving bar; and the lower plate support is vertically moved asthe level of the steps of the lower plate support moving bar broughtinto contact with the roller of the lower plate support is changed byturning or rotating the bar moving dial.

Further, a groove for temporarily restricting a motion of the lowerplate support may be formed on each of the steps.

Preferably, the lower plate support moving bar includes a lower platesupport shaking portion further extending from the step with the highestlevel and formed with a plurality of grooves.

The cell separation apparatus of the present invention may furthercomprise a housing, wherein the upper and lower plates are installedwithin the housing such that the bottom surface of the upper plate andthe cell mixture holding portion of the lower plate are positioned toface each other; the magnetic field applying means is positioned on thetop surface of the upper plate; the gap adjusting means is coupled withthe upper plate; and the upper plate is vertically moved by the gapadjusting means to adjust the gap between the upper and lower plates.

Preferably, the gap adjusting means further comprises a stopper forrestricting an upward motion of the upper plate support to allow thecell mixture layer to be maintained.

According to another aspect of the present invention for achieving theobject, there is provided a cell separation method comprising the stepsof (a) creating a cell mixture containing specific cells tagged withmagnetic beads into a cell mixture layer by adjusting a gap betweenupper and lower plates to be decreased such that the cell mixture whichis accommodated in a cell mixture holding portion of the lower plate inan upwardly convex shape can be adsorbed in a bottom surface of theupper plate positioned opposite to the cell mixture holding portion ofthe lower plate; (b) moving the specific cells toward the upper plate byapplying a magnetic field to the cell mixture layer created in step (a)from the upper plate and simultaneously moving cells other than thespecific cells toward the lower plate by means of gravity; and (c)allowing the specific cells moved toward the upper plate and the othercells moved toward the lower plate in step (b) to be divided and thenpositioned in the bottom surface of the upper plate and the cell mixtureholding portion of the lower plate, respectively, when the cell mixturelayer is separated by increasing the gap between the upper and lowerplates.

The cell separation method of the present invention may further comprisethe step of, after step (a), adjusting the gap between the upper andlower plates to maintain a thickness of the cell mixture layer at anoptimal cell separation state.

Further, the cell separation method of the present invention may furthercomprise the steps of (d1) creating a specific cell mixture layer bydecreasing the gap between the upper and lower plates after removing theother cells divided and positioned in the lower plate in step (c) orreplacing the lower plate with a new one and then injecting a buffersolution containing no cells in the lower plate; (e1) homogenizing thespecific cell mixture layer by changing the gap between the upper andlower plates repeatedly several time while maintaining the specific cellmixture layer created in step (d1) (f1) moving the specific cells towardthe upper plate by the magnetic field applied to the specific cellmixture layer homogenized in step (e1) from the upper plate andsimultaneously moving the other cells in the specific cell mixture layertoward the lower plate by means of gravity; and (g1) allowing thespecific cells moved toward the upper plate and the other cells movedtoward the lower plate in step (f1) to be divided and then positioned inthe bottom surface of the upper plate and the cell mixture holdingportion of the lower plate, respectively, when the specific cell mixturelayer is separated by increasing the gap between the upper and lowerplates.

Furthermore, the cell separation method of the present invention mayfurther comprise the steps of (d2) creating an other cell mixture layerby decreasing the gap between the upper and lower plates after removingthe specific cells divided and positioned in the upper plate in step (c)or replacing the upper plate with a new one and then additionallyinjecting a buffer solution containing no cells in the lower plate; (e2)homogenizing the other cell mixture layer by changing the gap betweenthe upper and lower plates repeatedly several times while maintainingthe other cell mixture layer created in step (d2) (f2) moving thespecific cells toward the upper plate by the magnetic field applied tothe other cell mixture layer homogenized in step (e2) from the upperplate and simultaneously moving the other cells in the other cellmixture layer toward the lower plate by means of gravity; and (g2)allowing the specific cells moved toward the upper plate and the othercells moved toward the lower plate in step (f2) to be divided and thenpositioned in the bottom surface of the upper plate and the cell mixtureholding portion of the lower plate, respectively, when the other cellmixture layer is separated by increasing the gap between the upper andlower plates.

ADVANTAGEOUS EFFECTS

According to the cell separation apparatus and method of the presentinvention, necessary cells can be separated through a process ofcreating a cell mixture layer by adjusting a vertical gap of the cellseparation chip composed of an upper plate and a lower plate foraccommodating a cell mixture and of adjusting a thickness of the createdcell mixture layer and separating the layer. Therefore, all the cellscan be separated using the same cell separation chip regardless of thesize of cells. Further, the separation process can be performed withoutany additional processes such as a pipetting process, a rotating processand a buffer solution injection process. Furthermore, separationefficiency can be enhanced by adjusting the thickness of the cellmixture layer in accordance with the separating conditions whileapplying a relatively strong magnetic field to the cells. Moreover, theseparation efficiency can be further enhanced by additionally removingunnecessary cells through the homogenization process performed after theseparation of the necessary cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of an apparatus for separatingcells by applying a magnetic field to a cell mixture layer in whichspecific cells tagged with magnetic beads are mixed according to anembodiment of the present invention.

FIG. 2 is a view showing a state where upper and lower plates are notmounted to the cell separation apparatus according to the embodiment ofthe present invention.

FIG. 3 is a view showing a state where a cell mixture is contained inthe lower plate of the cell separation apparatus according to theembodiment of the present invention.

FIG. 4 is a view showing a state where the cell mixture contained in thelower plate shown in FIG. 3 is formed into the cell mixture layer and anupper body is then covered onto a lower body in order to start theseparation of cells.

FIGS. 5 to 8 are views illustrating the operation of the cell separationapparatus and the state of the cell mixture when the cells are separatedin the cell separation apparatus according to the embodiment of thepresent invention.

FIG. 9 is a view showing the state of the upper and lower plates whenthe upper body has been separated from the lower body after the processof FIG. 8.

FIG. 10 is a view showing a state where cells other than the specificcells in the lower plate are removed and a new solution is then addedinto the lower plate to enhance the purity of the specific cellsremaining in the upper plate.

FIGS. 11 to 18 are views each illustrating a process of causing asolution of the specific cells to be in a homogeneous state and thenseparating the specific cells after the upper and lower bodies arecoupled with each other in a state of FIG. 10.

FIG. 19 is an exploded perspective view of an apparatus for separatingcells by applying a magnetic field to a cell mixture layer in whichspecific cells tagged with magnetic beads are mixed according to anotherembodiment of the present invention.

FIGS. 20 to 22 are views each illustrating a process of separating thecells using the cell separation apparatus according to anotherembodiment of the present invention.

FIG. 23 is a view showing a state where the specific cells remaining inthe upper plate are removed and a proper amount of a new solution isadded into the lower plate to enhance the purity of the lower othercells remaining in the lower the plate after the process of FIG. 22.

FIGS. 24 to 28 are views each illustrating a process of causing asolution of the other cells to be in a homogeneous state and thenseparating the other cells after the upper and lower bodies are coupledwith each other in a state of FIG. 23.

FIGS. 29 and 30 are perspective views showing an apparatus forseparating cells by applying a magnetic field to a cell mixture layer inwhich specific cells tagged with magnetic beads are mixed according to afurther embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

As shown in FIGS. 1 to 4, a cell separation apparatus 10 according to anembodiment of the present invention comprises an upper body 11 and alower body 12. Here, a process of separating cells from a cell mixtureis executed in a state where the upper body 11 for causing a magneticfield to be applied to the cell mixture is covered onto and thenintegrally coupled with the lower body 12 containing the cell mixture.

The upper body 11 includes an upper plate 111, magnets 113 positioned atan upper surface of the upper plate 111 to apply a magnetic field, anupper housing 115 with an open bottom for accommodating the upper plate111 and the magnets 113 therein, and upper plate fixing means 114 forfixing the upper plate 111 to the upper housing 115.

The upper plate 111 includes a plurality of cell mixture adsorbingportions 112 formed on a lower surface of the upper plate to adsorb acell mixture from the lower body 12. The lower plate 111 may be formedinto a flat and transparent chip with a thickness of 3 mm or less.

Each of the cell mixture adsorbing portions 112 may be formed eitherinto a recess when the upper plate 111 is made of a hydrophilicbiocompatible material such as polymethylmethacrylate (PMMA),polypropylene or polyimide or into a ring when the upper plate is madeof a hydrophobic biocompatible material such as polydimethylsiloxane(PDMS). In such a case, the cell mixture is adsorbed into the recess orthe circumference of the adsorbed cell mixture is enclosed by the ringto define the boundary of the cell mixture.

Each of the magnets 113 is installed in a state where they areaccommodated in the upper housing 115. Each of the magnets 113 ispreferably positioned to correspond to the cell mixture adsorbingportion 112 such that the magnetic field generated from the relevantmagnet can be concentrated on the relevant cell mixture adsorbingportion 112. In such a case, the size of the magnet 113 is equal to orslightly smaller than that of the cell mixture adsorbing portion and thethickness of the cell mixture adsorbing portion 112 is formed smallerthan that of the upper plate 111 with a thickness of 3 mm or less suchthat the magnetic field can be sufficiently exerted thereon.

In order to move specific cells tagged with magnetic beads in the cellmixture contained in the lower body toward the upper plate 111, themagnetic field generated in the magnet 113 should be maintained at acertain level enough to overcome the gravity exerted on the specificcells.

In the embodiments of the present invention, each of the magnets is madeof a neodymium permanent magnet such that it can generate a relativelystrong magnetic field with a strength of approximately 0.5 T whileoccupying a minimum space. Accordingly, the compact cell separationapparatus with high separation efficiency can be obtained.

The upper plate fixing means 114 allows the upper plate 111 to be fixedto the upper housing 115 in a state where an upper surface of the fixingmeans faces the magnet 113.

The lower body 12 includes a lower plate 121, a lower plate support 123for holding the lower plate 121 to perform the separation process insuch a state where the lower plate 121 is securely seated in the lowerplate support, a lower plate support moving dial 124 which rotates tocause the lower plate support 123 to move in a vertical direction, and alower housing 126 with an open top for accommodating the lower plate121, the lower plate support 123 and the lower plate moving dial 124therein.

Dial stopper 125, 125′ and 125″ for restricting the lower plate supportmoving dial from being rotated beyond a certain limit are installed atpredetermined positions on a bottom surface of the lower plate supportmoving dial 124 and corresponding positions on a floor surface of thelower housing 126.

The lower plate 121 is configured such that a cell mixture to beseparated is received in a cell mixture holding portion 122 formed in atop surface thereof in a convex shape. The lower plate 121 may be formedinto a flat and transparent chip with a thickness of 3 mm or less. Insuch a case, it is preferred that the lower plate be used together withthe upper plate 111 as a set of a cell separation chip and also bediscarded after one time use.

Each of the cell mixture holding portions 122 is shaped in a circular orsimilar form. A surface of the lower plate 121 is coated or recessedsuch that an angle of contact between a liquid surface and a solidsurface may be maintained at about 90 degrees or more when a liquid isplaced onto the level solid surface. Then, a certain amount of cellmixture is injected and received in the cell mixture holding portion 122such that the received cell mixture takes the shape of an upwardhemisphere.

Here, a case where the cell mixture holding portion 122 is formed into arecess will be discussed. If the lower plate 121 is made of ahydrophilic material, no additional treatment is required since the cellmixture does not flow out of the recess. However, if the lower plate 122is made of a hydrophobic material, an inner surface of the recess shouldbe coated with a hydrophilic material to allow the cell mixture to beeasily adsorbed.

The cell mixture holding portion 122 is formed at a positioncorresponding to the cell mixture adsorbing portion 112 of the upperplate 111 to create a layer while the cell mixture received in the cellmixture holding portion is adsorbed to the cell mixture adsorbingportion 112.

The layer of the adsorbed cell mixture is created into a space composedof a liquid to which magnetic field and gravity are applied upward anddownward, respectively. That is, the layer provides an environment inwhich the specific cells tagged with magnetic beads and the other cellsare clearly separated from each other by the opposite forces.

In a case where the cell mixture layer is formed between the cellmixture adsorbing portion 112 and the cell mixture holding portion 122to separate the cells, the cell separation process is performed while amaximum magnetic field is applied to the cell mixture adsorbing portion112 in a state where the cell mixture is not in a dynamic condition butin a static condition. Therefore, the cell separation process issimplified and its separation efficiency is increased. Further, sincethe cell mixture is consumed as much as required in the cell separation,the separation costs can also be reduced.

The amount of cells to be separated can be determined by adjusting thenumber or size of the cell mixture adsorbing portions 112 of the upperplate 111 and the cell mixture holding portions 122 of the lower plate121 which create the cell mixture layer between the portions. Inaddition, since several kinds of the cells can be separatedsimultaneously, the cell separation suitable to the variouscircumstances can be made.

However, as the volume of the cell mixture received in the cell mixtureholding portion 122, i.e. a surface area of the cell mixture holdingportion 122, is increased, the angle of contact is decreased due to anincreased effect of gravity.

Accordingly, in the embodiments of the present invention, an optimalcell separating environment for the easy cell separation is maintainedby keeping a diameter of the cell mixture holding portion 122 not morethan about 18 mm, preferably at a level of 14 mm such that the width ofthe cell mixture layer can be maintained within a range of 2 to 3 mm.

The lower plate support 123 is formed with a concave portion foraccommodating the lower plate 121 therein such that the cell separationprocess can be performed in a state where the lower plate 121 issecurely seated in the concave portion.

Further, the lower plate support 123 is installed to be movable withinthe lower housing 126 in a vertical direction. That is, a bolt-shapedconnection is formed on a bottom surface of the lower plate support 123to be engaged with a nut-shaped connection formed on the lower platesupport moving dial 124 such that the lower plate support and thus thelower plate 121 can be moved vertically by means of the rotation of thedial 124.

The lower plate support moving dial 124 is installed such the lowerplate support 123 can be moved vertically by rotating an exposed portionthereof in a horizontal direction in a state where a portion of the dialis exposed to the outside from a front side of the lower housing 126.

As the lower plate support moving dial 124 is moved horizontally, thelower plate support 123 is moved vertically to allow a vertical positionof the lower plate 121 to be adjusted. Accordingly, the cell mixturereceived in the cell mixture holding portion 122 is adsorbed into thecell mixture adsorbing portion 112 to either create a cell mixture layerbetween the upper and lower plates 111 and 121 or remove the createdcell mixture layer.

Here, in order to facilitate the cell separation process, a position orrange where the cell mixture layer is created or removed may beindicated on the lower plate support moving dial 124 using letters orcolors.

For example, numerals ‘1’, ‘2’ and ‘3’ are indicated on an edge of thelower plate support moving dial 124 in a counterclockwise direction insuch a manner that the numeral ‘1’ means a state where the cell mixturelayer is compressed, ‘2’ means a state where the cell mixture layer ismaintained at a proper width, and ‘3’ means a state where the cellmixture layer has been removed.

In the cell separation apparatus 10 according to an embodiment of thepresent invention, the width between the upper and lower plates 111 and121 can be set 2 mm for the ‘1’ state, 2.5 to 3 mm for the ‘2’ state,and 5 to 6 mm for the ‘3’ state when the diameter id the cell mixtureholding portion 122 is 14 mm.

Accordingly, in the cell separation apparatus 10 of the embodiment ofthe present invention, both the lower plate support 123 and the lowerplate support moving dial 124 perform a function as a means foradjusting the gap between the upper and lower plates 111 and 121 bymoving the lower plate 121 to execute a process of creating a cellmixture layer and adjusting the width of or separating the created cellmixture layer.

In a case where a cell mixture layer is created or the created layer isdivided to separate the cells while the gap between the upper and lowerplates 111 and 121 is adjusted to be increased or decreased, the cellseparation can be smoothly obtained without any additional equipmentsince the cell distribution due to the magnetic field and gravity in thelayer and the creation or separation of the layer can be simultaneouslydetermined.

Further, the dial stopper 125 is installed in the form of a protrusionat the ‘1’ and ‘3’ positions on the bottom surface of the lower platesupport moving dial 124.

In addition, the dial stopper 125′ formed on the floor surface of thelower housing 126 is installed in the form of a protrusion at a positionsuch that it is brought into contact with the ‘3’-position dial stopper125 and thus not further rotated when the numeral ‘1’ is exposed to theoutside from the front side of the lower housing 126 and that it isbrought into contact with the ‘1’-position dial stopper 125 and thus notfurther rotated when the numeral ‘3’ is exposed to the outside.

Therefore, the lower plate support moving dial 124 is rotated onlywithin the ‘1’ and ‘3’ positions by the dial stoppers 125 and 125′ suchthat a process of creating and separating the cell mixture layer can beconveniently performed without paying specific attention thereto.

The lower housing 126 is integrally coupled with the upper housing 115in such a manner that the open top of the lower housing is connected tothe open bottom of the upper housing. Some protrusions are formed aroundan outer rim of the lower housing 126 such that the lower housing can beeasily coupled with the upper housing 115.

When the lower and upper housings 126 and 115 are coupled with eachother, a closed space is defined between the housings such that anoptimal environment in which cells are alive is maintained whilemoisture evaporation or temperature change in the cell mixture layer isminimized.

Furthermore, in a case where the cells other than the specific cells aretagged to the separated specific cells within the cell separationapparatus 10 according to the embodiment of the present invention, thepurity of the specific cells can be further increased by separating andeliminating the other cells from the separated cells during thehomogenization process in a state where the cell mixture layer ismaintained.

A further dial stopper 125″ is installed at a front side of the lowerplate 126 such that the lower plate support moving dial 124 can berotated only within a limit where the layer is maintained when thehomogenization process is performed.

The dial stopper 125″ can be installed to move leftward or rightward.Thus, a portion of the dial stopper protruding toward the lower platesupport moving dial 124 is also moved leftward or rightward within thelower housing 126.

Therefore, if the dial stopper 125″ is moved leftward, the ‘1’-positiondial stopper 125 is hindered by the protruding portion of the dialstopper 125″ from being further rotated when the dial stopper 125 ispositioned such that the numeral ‘2’ is exposed to the outside. Thus,the homogenization process can be rapidly and conveniently performedwhile the cell mixture layer is maintained.

However, if the dial stopper 125″ is moved rightward, the ‘1’-positiondial stopper 125 is not hindered by the protruding portion of the dialstopper 125″ from being rotated and the lower plate support moving dial124 can thus be turned up to the ‘3’ position in a counterclockwisedirection even when the dial stopper 125 is positioned such that thenumeral ‘2’ is exposed to the outside.

Considering that the upper and lower plates 111 and 121 are newlyexchanged whenever cells are separated, the cell separation apparatus 10of the embodiment of the present invention may be divided into a cellseparation chip composed of the upper and lower plates 111 and 121, anda cell separation executing unit for adjusting the gap between the upperand lower plates 111 and 121 and executing the cell operation by meansof the magnetic field applied between the plates.

A process of separating cells from a cell mixture by using the cellseparation apparatus according to the embodiment of the presentinvention will be described.

As shown in FIG. 3, the upper body 11 of the cell separation apparatus10 of the present invention is first disconnected from the lower body12. Then, a cell mixture in which the specific cells tagged withmagnetic beads are mixed is injected and received in the lower plate 121of the lower body 12, i.e. each cell mixture holding portion 122 with adiameter of 14 mm, at an amount of 500

.

As shown in FIG. 4, the upper body 11 is covered onto the lower body 12such that they are integrally coupled with each other. Here, in a casewhere the numeral ‘1’ indicated on the lower plate support moving dial124 of the lower body 12 is positioned to be exposed to the outside asshown in FIG. 5, the ‘3’-position dial stopper 125 is brought intocontact with the dial stopper 125′ installed on the floor surface of thelower housing 126, and thus, the lower plate support moving dial 124cannot be further rotated.

In such a case, the cell mixture layer in which the specific cells andthe other cells are uniformly distributed between the cell mixtureadsorbing portion 112 of the upper plate 111 and the cell mixtureholding portion 122 of the lower plate 121 in a compressed state.

Next, if the lower plate support moving dial 124 positioned with thenumeral ‘1’ exposed to the outside is rotated in a counterclockwisedirection to cause the numeral ‘2’ to be exposed to the outside, thedial stopper 125 installed on the bottom surface of the lower platesupport moving dial 124 is moved rightward but is not brought intocontact with the dial stopper 125′ installed on the floor surface of thelower housing 126.

In such a case, the lower plate 121 is moved downward to therebyincrease the gap between the lower and upper plates such that thecompressed cell mixture layer can be formed into a layer with apredetermined width suitable for the cell separation.

However, it is not always preferred that the thickness of the cellmixture layer created between the upper and lower plates 111 and 121 bekept constant whenever cells are separated.

In general, as the vertical width of the cell mixture layer, i.e. thegap between the upper and lower plates 111 and 121 is increased, thecell separation can be clearly performed by means of the magnetic fieldapplied to the specific cells and the gravity applied to the othercells.

However, the compatibility (generality) that all the characteristics(size, density, degree of magnetization and the like) of the magneticbeads varying according to the manufacturers can be accepted after thecell mixture layer has been created should be obtained, or thecharacteristics of specific cells themselves should be properlycontrolled since the degrees of the specific cells contained in the cellmixture (cell concentration) are different from each other. Further, anadsorption degree may vary according to whether the material of theupper and lower plate 111 and 121 as a cell separation chip ishydrophilic or hydrophobic. Thus, the gap between the upper and lowerplates 111 and 121 should be adjusted to maintain the cell separation inan optimal state.

Here, if it is assumed that the optimal cell separation state can bemaintained by slightly increasing the width of the cell mixture layer socreated, the lower plate support moving dial 124 is slightly furtherrotated in a counterclockwise direction as compared with when thenumeral ‘2’ is exposed to the outside as shown in FIG. 7. That is, thelower plate 121 can be moved downward slightly further than as shown inFIG. 6.

In such a case, the dial stopper 125 installed on the bottom surface ofthe lower plate support moving dial 124 is further moved in acounterclockwise direction but is not yet brought into contact with thedial stopper 125′ installed on the floor surface of the lower housing126.

Then, if the current state is maintained for 7 to 15 minutes while themagnetic field is applied to the cell mixture layer with an optimalwidth through the magnets 113 disposed on the top surface of the upperplate 111, the specific cells in the cell mixture layer are moved towardthe upper plate 111. At the same time, the other cells in the cellmixture layer are also moved toward the lower plate 121 by means ofgravity.

Finally, if the lower plate support moving dial 124 is turned in acounterclockwise direction with the numeral ‘3’ exposed to the outside,the ‘1’-position dial stopper 125 is also moved in the counterclockwisedirection and is brought into contact with the relevant dial stopper125′ installed on the floor surface of the lower housing 126. Thus, itis possible to prevent the lower plate support moving dial 124 frombeing further rotated.

In such a case, the lower plate 121 will be completely moved downwardsuch that the cell mixture layer can be separated. As a result, thespecific cells and the other cells are divided and then positioned inthe upper and lower plates 111 and 121, respectively.

Therefore, the dial stopper 125 and 125′ perform the functions ofcreating the cell mixture layer, adjusting the width of the created cellmixture layer, separating the layer and separating the cells in aconvenient way while being turned within the range of the ‘1’ and ‘3’positions.

As shown in FIG. 9, the specific cells and the other cells are dividedand then received in the upper plate 111 of the upper body 11 and thelower plate 121 of the lower body 12, respectively. Thereafter,necessary cells are collected and then utilized.

As shown in FIG. 4, when the upper body 11 is covered onto the lowerbody 12 such that they are integrally coupled with each other, the lowerplate support moving dial 124 of the lower body 12 is positioned in astate where the numeral ‘2’ or ‘3’ is exposed to the outside.Preferably, the lower plate support moving dial 124 is turned in aclockwise or counterclockwise direction to maintain or create the cellmixture layer.

According to the present invention, the cell separation apparatus 10 canbe simply operated and be portable due to the small size thereof.Further, specific cells that should be maintained at a temperature of 4°C. can be simply stored and then separated in a refrigerator withoutmoving the cell separation apparatus containing the specific cells to aspecific place or using additional cooling equipment.

The cells adsorbed to the upper plate 111 should become the specificcells. In fact, however, the other cells may be adsorbed to the upperplate 111 when the cell mixture layer is created. Alternatively, aportion of the other cells adhering to the specific cells may beattracted together with the specific cells when they are attractedtoward the upper plate 111 by means of the magnetic field generated fromthe magnets 113. Therefore, unnecessary cells other than the specificcells adsorbed to the upper plate 111 should also be separated.

Accordingly, in order to create an environment in which the other cellsadhering to the specific cells can be optimally separated, the separatedother cells are removed from the lower plate 121 or the used lower plateis replaced with a new lower plate.

Then, a buffer solution containing no cells is injected and accommodatedin the lower plate 121 as shown in FIG. 10. Finally, the upper body 11having the upper plate 111 with the specific cells adsorbed therein iscovered onto the lower body 12 having the lower plate 121 such that thetwo bodies are integrally coupled with each other.

As shown in FIG. 11, since the lower plate support moving dial 124 ispositioned such that the numeral ‘3’ can be exposed to the outside, thespecific cells are positioned in the upper plate 111 and the buffersolution is positioned in the lower plate 121, but a cell mixture layeris not yet created between the two plates.

Next, as shown in FIGS. 12 and 13, if the lower plate support movingdial 124 positioned at the ‘3’ position is sequentially turned to the‘2’ and ‘1’ positions in a clockwise direction, the dial stopper 125installed on the bottom surface of the lower plate support moving dial124 is moved in a clockwise direction and then brought into contact withthe dial stopper 125′ installed on the floor surface of the lowerhousing 126 such that the lower plate support moving dial 124 cannot befurther turned in a clockwise direction.

In such a case, as the lower plate 121 is moved upward, the gap betweenthe upper and lower plates is decreased. Thus, the specific cellsadsorbed in the upper plate 111 and the buffer solution contained in thelower plate 121 are mixed to create a specific cell mixture layersuitable for the cell separation such that the created layer is again ina vertically compressed state.

Further, as shown in FIG. 14, while the lower plate support moving dial124 is still maintained at the ‘1’ position, the dial stopper 125″ ismoved leftward.

Furthermore, as shown in FIG. 15, if the lower plate support moving dial124 positioned at the ‘1’ position is turned in a counterclockwisedirection to be positioned at the ‘2’ position after the dial stopper125″ has been moved leftward, the dial stopper 125″ prevents the dialstopper 125 positioned at the ‘1’ position from being rotated in acounterclockwise direction such that the lower plate support moving dial124 cannot be further turned in the counterclockwise direction.

Next, the processes illustrated in FIGS. 15 and 16 are repeated severaltimes, e.g. 10 times, while maintaining the specific cell mixture layercreated between the upper and lower plates 111 and 121 to rapidly andconveniently homogenize the specific cell mixture layer.

Then, the specific cell mixture layer is shaken vertically to the utmostin a state where the specific cell mixture layer is maintained, andconsequently, the other cells that adhere to the upper plate 111together with the specific cells reacting with the magnets 113 areseparated such that the layer can be in a single cell state.

After the homogenization process has been completed, the specific cellmixture layer is left alone for 7 to 15 minutes in a state where thenumeral ‘2’ is exposed to the outside, as shown in FIG. 15. Then, thespecific cells in the homogenized cell mixture layer are moved towardthe upper plate 111 by means of the magnetic field applied theretothrough the magnets 113, whereas the other cells in the cell mixturelayer are moved toward the lower plate 121 by means of gravity.

If the dial stopper 125″ is moved rightward and the lower plate supportmoving dial 124 is then turned in the counterclockwise direction towardthe ‘3’ position in such a state as shown in FIG. 17, the ‘1’-positiondial stopper 125 is moved rightward and brought into contact with thedial stopper 125′ installed on the floor surface of the lower housing126 to restrict the rotation of the lower plate support moving dial 124.

In such a case, the lower plate 121 has completely will be completelymoved downward such that the cell mixture layer can be separated. As aresult, the specific cells and the other cells are divided and thenpositioned in the upper and lower plates 111 and 121, respectively.

That is, the other cells contained in the separated specific cells areadditionally separated and removed from the upper plate 111 to therebymarkedly enhance the purity of the necessary specific cells.

FIG. 19 shows a cell separation apparatus 20 according to anotherembodiment of the present invention. The cell separation apparatus shownin FIG. 19 is identical to the cell separation apparatus 10 of theprevious embodiment except their gap adjusting means for adjusting thegap between the upper and lower plates. Hereinafter, the gap adjustingmeans for adjusting the gap between the upper and lower plates will bemainly described.

In the cell separation apparatus 10 according to the previous embodimentof the present invention, the lower plate support moving dial 124 whichserves as a gap adjusting means for adjusting the gap between the upperand lower plates and includes the nut-shaped connection engaged with thebolt-shaped connection formed on the bottom surface of the lower platesupport 123 is rotated to vertically move the lower plate support 123such that the vertical position of the lower plate 121 can be adjusted.On the other hand, in the cell separation apparatus 20 according to thisembodiment of the present invention, a bar moving dial 227 having apinion gear portion meshed with a rack portion formed on a side surfaceof a lower plate support moving bar 224 is rotated to horizontally movethe lower plate support moving bar 224 with three steps formed on anupper surface of the lower plate support moving bar 224 such that alower plate support 223 can be moved vertically by using a roller formedon a bottom surface of the lower plate support 223.

The three steps in the cell separation apparatus 20 according to thisembodiment of the present invention perform the same function as the‘1’, ‘2’ and ‘3’ positions indicated on the edge of the lower platesupport moving dial 124 of the cell separation apparatus 10 according tothe previous embodiment of the present invention.

The lower plate support 223 includes a roller, unlike the bolt-shapedconnection formed on the lower plate support 123 of the cell separationapparatus 10 according to the previous embodiment of the presentinvention.

The lower plate support moving bar 224 is formed with three steps whoselevels are decreased from left to right to implement the vertical motionof the lower plate support 223 in correspondence with the ‘1’ to ‘3’positions. However, the number of steps may be changed, or the steps maybe connected in an inclined manner.

The roller formed below the lower plate support 223 is kept in contactwith any one of the three steps formed on the upper surface of the lowerplate support moving bar 224.

Therefore, if the lower plate support moving bar 224 is moved in ahorizontal direction along its longitudinal axis, the position of thesteps brought into contact with the roller is changed to vertically movethe lower plate support 223 including the roller.

Grooves 225 for temporarily restricting the motion of the lower platesupport 223 are formed on the three steps, respectively.

The grooves 225 cause the roller to be temporarily held therein until apre-determined force is applied to the roller. Thus, the aforementionedpositions ‘1’, ‘2’ and ‘3’ can be sensibly divided such that a user caneasily create the layer, adjust the width of the created layer andseparate the layer.

Further, a lower plate support shaking portion 226 extending from thegroove 225 and formed with a plurality of grooves is installed on theupper surface of the lower plate support moving bar 224.

The vibration generated from the lower plate support shaking portion 226is transmitted to the lower plate 221 through the lower plate support223 to perform a process of homogenizing a cell mixture layer containingcells other than the specific cells.

The bar moving dial 227 is configured in such a manner that a portion ofits edge is exposed to the outside from a front side of a lower housing228. If the exposed portion of the bar moving dial is turned in acounterclockwise direction, the lower plate support moving bar 224 ismoved from right to left.

At this time, the pinion gear portion of the bar moving dial 227 isthreadedly engaged with the rack portion of the lower plate supportmoving bar 224. That is, as the bar moving dial 227 is turned, the lowerplate support moving bar 224 is moved horizontally. Thus, the level ofthe steps brought into contact with the roller of the lower platesupport 223 is changed to allow the lower plate support 223 to bevertically moved.

A process of separating cells using the cell separation apparatus 20according to this embodiment of the present invention so configured willbe explained as follows.

A cell mixture is first injected and received in a lower body 22 of thecell separation apparatus according to this embodiment of the presentinvention, and an upper body 21 is then covered onto the lower body 22such that the two bodies can be integrally coupled with each other. Asshown in FIG. 20, if the bar moving dial 227 is positioned such that thenumeral ‘1’ is exposed to the outside, the roller of the lower platesupport 223 is temporarily fixed and positioned in the first groove225-1 corresponding to the ‘1’ position.

In such a case, a cell mixture layer in which the specific cells and theother cells are uniformly distributed is created in a compressed statebetween a cell mixture adsorbing portion 212 of an upper plate 211 and acell mixture holding portion 222 of the lower plate 221.

Next, if the bar moving dial 227 is turned in the counterclockwisedirection from the ‘1’ position to another position where the numeral‘2’ is exposed to the outside, the lower plate support moving bar 224 ismoved leftward to thereby move the lower plate support 223 downward andto increase a gap between the upper and lower plates as shown in FIG.21. Therefore, the cell mixture layer with a width suitable for the cellseparation can be created.

In such a case, the roller of the lower plate support 223 is kept in astate where it is temporarily fixed into the second groove 225-2corresponding to the ‘2’ position.

Then, if the current state is maintained for 7 to 15 minutes while themagnetic field is applied to the cell mixture layer with an optimalwidth through the magnets 213 disposed on the top surface of the upperplate 211, the specific cells in the cell mixture layer are moved towardthe upper plate 211. At the same time, the other cells in the cellmixture layer are also moved toward the lower plate 221 by means ofgravity.

Finally, if the bar moving dial 227 is turned in the counterclockwisedirection from the ‘2’ position to another position where the numeral‘3’ is exposed to the outside, the lower plate support moving bar 224 ismoved leftward to thereby to completely move the lower plate support 223downward and to increase the gap between the upper and lower plates, asshown in FIG. 22. Thus, the cell mixture layer is separated such thatthe specific cells and the other cells are divided and then positionedin the upper and lower plates 211 and 221, respectively.

In such a case, the roller of the lower plate support 223 is kept in astate where it is temporarily fixed into the third groove 225-3corresponding to the ‘3’ position.

Accordingly, as shown in FIG. 9, since the specific cells and the othercells are divided and then positioned in the upper and lower plates 211and 221, respectively, necessary cells will be collected and utilizedlater.

In the cell separation apparatus 20 according to this embodiment of thepresent invention, the steps of creating the cell mixture layer,adjusting the thickness of the later and separating the layer can besensibly divided at temporarily fixed conditions such that a user canfeel the conditions and perform the convenient and precise cellseparation process.

The cells adsorbed to the lower plate 221 should become the cells otherthan the specific cells. In fact, however, a portion of the specificcells adhering to the other cells may be attracted together with theother cells when they are attracted toward the lower plate 221 by meansof gravity.

Here, the cells to be collected generally become the specific cellstagged with magnetic beads. However, if they are clinically utilized,the other cells with no magnetic beads tagged thereto may be employed.

Accordingly, the cells other than unnecessary specific cells adsorbingto the upper plate 211 should be separated.

In order to create an environment where the specific cells adhering tothe other cells can be optimally separated, the separated specific cellsshould be removed from the lower plate 121 or the used lower plate isreplaced with a new lower plate as shown in FIG. 23.

Further, the upper body 21 including the empty upper plate 211 iscovered onto the lower body 22 including the lower plate 221 injectedwith a buffer solution with no cells contained therein at an amountcorresponding to a degree that the specific cells transferred to theupper plate 211 are replenished, such that the upper and lower bodiescan be integrally coupled with each other.

In such a case, as shown in FIG. 24, the bar moving dial 227 ispositioned in a state where the numeral ‘3’ is exposed to the outside.That is, no cell mixture layer is created in a state where the upperplate 211 is only empty.

As shown in FIGS. 25 and 26, if the bar moving dial 227 is turned in aclockwise direction from the ‘3’ position through the ‘2’ position tothe ‘1’ position, the lower plate support moving bar 224 is movedrightward and the roller of the lower plate support 223 is positioned onthe highest step. Thus, the lower plate support 223 can be pushedupward.

In such a case, as the lower plate 221 is moved upward, the gap betweenthe upper and lower plates is decreased such that the cell mixture ofthe other cells in the lower plate 221 absorbs against the upper plate211 to create the mixture layer of the other cells suitable for the cellseparation. Then, the created layer is in a compressed state.

Next, as shown in FIG. 26, if the bar moving dial 227 is further turnedfrom the ‘1’ position in the clockwise direction (the lower platesupport 223 is moved at a dotted line of FIG. 26), the vibrationgenerated when the roller of the lower plate support 223 travelsrightward along the lower plate support shaking portion 226 in a statewhere they are brought into contact with each other is transmitted tothe lower plate 221 through the lower plate support 223.

At this time, even when the bar moving dial 227 is turned in thecounterclockwise direction to move the roller of the lower plate support223 rightward up to the first groove 225-1 corresponding to the ‘1’position, the vibration generated when the roller of the lower platesupport 223 travels leftward along the lower plate support shakingportion 226 in a state where they are brought into contact with eachother is transmitted to the lower plate 221 through the lower platesupport 223.

The homogenization process is performed while the vibration transmittedto the lower plate 221 is applied to the other cell mixture layer. Thisprocess is repeated several times, e.g. less than 10 times, whilemaintaining the other cell mixture layer created between the upper andlower plates 211 and 221.

Then, since a maximum vibration can be applied to the maintained othercell mixture layer, the specific cells adhering to lower plate 221together with the other cells fallen onto the lower plate 221 by meansof gravity come off from the lower plate 221. Subsequently, the specificcells are moved toward the upper plate 211 by means of the magneticfield applied thereto from the magnets 213.

After the homogenization process has been performed, the cell mixturelayer is left alone for 7 to 15 minutes in a state where the roller istemporarily fixed in the second groove 225-2 by positioning the barmoving dial at the ‘2’ position, as shown in FIG. 27. As a result, thespecific cells are moved toward the upper plate 211 by means of themagnetic field applied thereto from the magnets 213 while the othercells are moved toward lower plate 221 by means of gravity.

In such a state, if the bar moving dial 227 is further turned in thecounterclockwise direction from the ‘2’ position to the ‘3’ position,the lower plate support moving bar 224 is moved leftward and thus thelower plate support 223 is completely moved downward as shown in FIG.28. Therefore, the gap between the upper and lower plates are increasedto thereby separate the cell mixture layer such that the specific cellsand the other cells are divided and then received in the upper and lowerplates 211 and 221, respectively.

At this time, the roller of the lower plate support 223 is kept in astate where it is temporarily fixed in the third groove 225-3 forrestricting the motion of the lower plate support.

Accordingly, the unnecessary specific cells can be additionallyseparated from the other cell mixture from which the specific cells havebeen previously separated and finally removed from the lower plate 221.Thus, the purity of the other cells can be greatly enhanced.

A cell separation apparatus 30 according to a further embodiment of thepresent invention is identical to those of the previous embodiments ofthe present invention except the following points. That is, in theprevious embodiments of the present invention, the upper body to whichthe magnetic field is applied is covered onto the lower body in whichthe cell mixture is contained such that the two bodies are integrallycoupled with each other. At this state, the lower plate is moved toadjust the gap between the upper and lower plates such that the specificand other cells are separated. On the other hand, in this embodiment ofthe present invention, the upper and lower plates are installed in asingle housing and the upper plate is then moved to adjust the gapbetween the upper and lower plates such that the specific and othercells are separated. Hereinafter, the foregoing difference will bemainly described.

In the cell separation apparatus 30 according to the further embodimentof the present invention, an upper plate support 34 and a lower platesupport 36 are installed to be movable in a fore and aft directionwithin a housing 31 with a portion thereof opened forward. Here, thelower plate support 36 is fixed installed at a lower portion of thehousing 31, whereas the upper plate support 34 is positioned above thelower plate support 36 by a certain distance and installed to bevertically movable within the housing 31.

The upper plate support 34 and the lower plate support 36 are formedwith recesses in which an upper plate 33 and a lower plate 35 arereceived, respectively. That is, the upper and lower plates 33 and 35are installed within the recesses, respectively, such that cell mixtureadsorbing portions and cell mixture holding portions face each other.

Further, a magnet 32 is installed above the upper plate support 34 toface a top surface of the upper plate 33.

In addition, the upper plate support 34 is threadedly engaged (notshown) with an upper plate support moving dial 37 installed on an outerside of the housing 31 such that it can be vertically moved by means ofthe rotation of the upper plate support moving dial 37.

Here, when the upper plate support 34 is vertically moved, both themagnet 32 and the upper plate 33 are moved together with the support 34.

Further, a dial stopper 38 is installed on another outer side of thehousing 31 to restrict the vertical motion of the upper plate support 34which is vertically moved as the upper plate support moving dial 37 isturned or rotated.

The dial stopper 38 restricts the upward motion of the upper platesupport 34 by using a protrusion formed to protrude into the housing 31when the dial stopper is pushed, such that a cell mixture layer can bemaintained.

A process of separating cells using the cell separation apparatusaccording to the further embodiment of the present invention soconfigured will be hereinafter described.

First, the lower plate support 36 is taken out in a front loading modetoward a user standing in front of the cell separation apparatus and thecell mixture is then injected and contained in the lower plate 35 in astate where the lower plate 35 is securely seated in the recess of thelower body with the cell mixture holding portions facing upward. Then,the lower plate support 36 is again pushed rearward into the housing 31.

Next, the upper plate support 34 is taken out in a front loading modetoward the user standing in front of the cell separation apparatus andthe upper plate 33 is securely seated in the recess of the upper bodywith the cell mixture adsorbing portions facing upward. Theaforementioned processes may be performed in a reverse order.

Then, the magnet 32, the upper plate 33 and the upper plate support 34are vertically moved together with one another as the upper platesupport moving dial 37 is turned. Thus, the upper plate 33 is movedtoward the lower plate 35 to be closer to each other to create the cellmixture layer. Then, the magnetic field is applied to the created cellmixture layer from the upper plate 33 using the magnet 32 to move thespecific cells and the other cells toward the upper plate 33 and thelower plate 35, respectively. Finally, the upper plate 33 is again movedfar away from the lower plate 35 to allow the cell mixture layer to beseparated.

Here, if the upper plate support 34 and the lower plate support 36 arebrought into contact with each other, the cell mixture layer can becreated in a compressed state between the upper and lower plates 33 and35.

Finally, the specific cells adsorbed in the upper plate 33 can berecovered after the upper plate support 34 is pulled out, or the othercells in the lower plate 35 can be recovered after the lower platesupport 36 is pulled out.

Further, the homogenization process performed in the cell separationapparatus 10 and 20 according to the previous embodiments of the presentinvention may be executed by operating the dial stopper 38.

The process of separating cells from a cell mixture according to theembodiments of the present invention was tested as follows.

A cell mixture obtained by flushing bone marrow cells is first labeledwith magnetic beads. The concentration of the cell mixture used toseparate specific cells from the cell mixture for each antibody is 10⁷/

, and the cell separation process is performed in a cold chamber at atemperature below 4° C. to exclude the temperature effects.

In case of the cell separation apparatus 10 and 20 according to theembodiments of the present invention, since the upper and lower bodiesare coupled integrally with each other, a closed space is created andthe cell mixture is injected in the cell mixture holding portions. Then,if the cell separation apparatus including the cell mixture is placedfor 7 to 15 minutes within a refrigerator or the like of which insidetemperature is kept at about 4° C. in a state where the lower platemoving dial is placed at the ‘3’ position, the cell separation processis fully completed without any additional operations or cold chamber.Thus, it is very convenient to perform the cell separation process.

When it is defined that the separated specific cells are positive whilethe other cells are Negatives, the results of the separation efficiencyof positive obtained using a variety of antibodies can be expressed asfollows.

TABLE 1 Content of Specific Antibody Cells Contained in Separation UsedControl Efficiency CD45 80% 97.5% Ter119 13.3%   98% Sca1 2.09%   28%SSEA1 <1% 19.5% SSEA3 <1% 13%

Further, after the cells other than the separated specific cells, i.e.negative, have been collected, a separation error ratio (a ratio thatthe positive remains in the negative in a state where the positive isnot fully separated) is confirmed. The results are expressed as follows.

TABLE 2 Content of Specific Antibody Cells Contained in Separation UsedControl Efficiency CD45 80% 5.4% Ter119 13.3%   18.3% Sca1 2.09%   1.8%SSEA1 <1% 0.8% SSEA3 <1% 0.5%

The process of enhancing the purity of cells separated by applying amagnetic field to a cell mixture according to the embodiments of thepresent invention was specifically tested as follows.

In an experiment example of the present invention, the state of cellswas measured from the negative solution collected after the cellseparation process such that the error ratio can be confirmed bychecking how much the positive is contained in the solution in the lowerplate, i.e. the negative solution.

Basically, the cells used in the experiment example are cells reactingwith the Ter119 antibody, the magnetic field from the magnet is 0.5 T,and the exposed period of time is 9 minutes.

As seen from Table 3, the separation efficiency when the specific cellsexist (positive exists) in the cell mixture at a content level of 13.3%(Control.002 means a control and is used to compare the state of cellsand the separation efficiency by measuring the state of cell mixturebefore the test) is expressed as follows.

As seen from Table 4, MB-P.002 corresponds to the measurement of thestate of positive after the separation which indicates a separationefficiency of 93.77%. At the same time, MB1-N.023 shows that thepositive is contained in the negative at a content level of 10.10%.

Here, the process of relatively enhancing the purity of the negative byremoving the positive from the negative solution containing 10.10% ofthe positive was performed. It was also confirmed that an error ratiowas decreased from an initial ratio (i.e., 10.10%) to 8.01% whenexamined under the assumption that the positive in the negative fractionis considered as an error.

Accordingly, it was confirmed that the positive contained in thenegative solution as an error was decreased by about 20.7% and thus thepurity of the negative was increased by about 2.3%.

In the case of the positive, cells reacting with the Sca1 antibody weretested under the same conditions (i.e., exposure to the magnetic fieldof 0.5 T for 9 minutes).

TABLE 5 Positive Negative (Purity En- (Positive Positive hancement)Error) Control 2.09 Cell Separation 6.30% 13.55% 2.51% Apparatusaccording to Present Embodiments

As shown in Table 5, when the cell mixture (control) containing 2.09% ofthe positive are separated, it was confirmed that the content of thepositive is 6.30% and the content of the negative is 2.51%.

Here, since the content of the specific cells is as low as 2.09%, theprocess of enhancing the purity of the positive was additionallyperformed. Thus, it was confirmed that the content of the positive wasincreased to 13.55% corresponding to about 115% increase as comparedwith the initial content.

The present invention is not limited to the aforementioned embodimentsof the present invention. Various modifications and changes can be madethereto within the scope of the present invention defined in theappended claims. However, the changes and modifications should beconstrued as falling within the scope of the present invention.

Although it has been described in the embodiments of the presentinvention that the upper or lower plate is moved directly by the user tocreate the cell mixture layer, adjust the width of the created layer andseparate the layer, a driving means such as a motor and a microprocessorfor controlling the motor may be utilized to automatically operate thecell separation apparatus based on the predetermined algorithm insteadof using the manual operation of the user.

INDUSTRIAL APPLICABILITY

According to the cell separation apparatus and method of the presentinvention, the necessary cells can be separated through the process ofcreating the cell mixture layer, adjusting the thickness of the layerand separate the layer by adjusting the vertical gap between the upperand lower plates of the cell separation chip which comprises the upperplate and the lower plate containing the cell mixture. Further, afterthe necessary cells have been separated, the homogenization process canbe performed to additionally remove unnecessary cells.

1. A cell separation apparatus, comprising: a lower plate provided with a cell mixture holding portion, in which a cell mixture containing specific cells tagged with magnetic carriers is accommodated in an upwardly convex shape, at a top surface thereof; an upper plate positioned above the lower plate to face each other and to adsorb the cell mixture accommodated in the cell mixture holding portion of the lower plate into a bottom surface thereof; a magnetic field applying means positioned on a top surface of the upper plate; and a gap adjusting means coupled to the upper or lower plate to adjust a gap between the upper and lower plates to be increased or decreased, wherein the gap between the upper and lower plates is decreased by the gap adjusting means such that the cell mixture accommodated in the cell mixture holding portion is adsorbed in the bottom surface of the upper plate and then formed into a cell mixture layer, and the gap between the upper and lower plates is increased by the gap adjusting means such that the specific cells moved toward the upper plate by means of a magnetic field applied to the created cell mixture layer through the magnetic field applying means and cells other than the specific cells moved toward the lower plate by means of gravity are divided and then positioned in the bottom surface of the upper plate and the cell mixture holding portion of the lower plate, respectively.
 2. The apparatus as claimed in claim 1, wherein a cell mixture adsorbing portion is formed at the bottom surface of the upper plate such that the cell mixture holding portion of the lower plate is positioned to correspond to the cell mixture adsorbing portion of the upper plate.
 3. The apparatus as claimed in claim 1, further comprising an upper housing with an open bottom and a lower housing with an open top, wherein the magnetic field applying means and the upper plate are installed within the upper housing such that the magnetic field applying means is positioned on the top surface of the upper plate, the lower plate and the gap adjusting means are installed within the lower housing such that the gap adjusting means is coupled to the lower plate, and the lower plate is vertically moved by the gap adjusting means to adjust the gap between the upper and lower plates in a state where the upper housing is coupled with the lower housing such that the bottom surface of the upper plate and the cell mixture holding portion of the lower plate are positioned to face each other.
 4. The apparatus as claimed in claim 2, wherein the gap adjusting means comprises a lower plate support formed with a recess for accommodating the lower plate therein at a top side thereof and a bolt-shaped connection at a bottom side thereof, and a lower plate support moving dial having a nut-shaped connection threadedly engaged with the bolt-shaped connection of the lower plate support; and the lower plate support is vertically moved by turning or rotating the lower plate support moving dial.
 5. The apparatus as claimed in claim 4, wherein the gap adjusting means further comprises a dial stopper for restricting the rotation of the lower plate support moving means such that the cell mixture layer can be maintained.
 6. The apparatus as claimed in claim 5, wherein the dial stoppers are installed on a bottom surface of the lower plate support moving dial and a predetermined portion of the lower housing such that the dial stopper is brought into contact with the bottom surface of the lower plate support moving dial at a position where the rotation of the lower plate support moving dial should be prevented.
 7. The apparatus as claimed in claim 3, wherein the gap adjusting means comprises a lower plate support formed with a recess for accommodating the lower plate therein at a top side thereof and a roller at a bottom side thereof, a lower plate support moving bar for vertically moving the lower plate support in such a manner that the roller of the lower plate support is brought into contact with a plurality of steps with different levels decreasing from one side to another side, and bar moving dial having a pinion portion meshed with a rack portion formed on a side surface of the lower plate support moving bar; and the lower plate support is vertically moved as the level of the steps of the lower plate support moving bar brought into contact with the roller of the lower plate support is changed by turning or rotating the bar moving dial.
 8. The apparatus as claimed in claim 7, wherein a groove for temporarily restricting a motion of the lower plate support is formed on each of the steps.
 9. The apparatus as claimed in claim 8, wherein the lower plate support moving bar includes a lower plate support shaking portion further extending from the step with the highest level and formed with a plurality of grooves.
 10. The apparatus as claimed in claim 1, further comprising: a housing, wherein the upper and lower plates are installed within the housing such that the bottom surface of the upper plate and the cell mixture holding portion of the lower plate are positioned to face each other; the magnetic field applying means is positioned on the top surface of the upper plate; the gap adjusting means is coupled with the upper plate; and the upper plate is vertically moved by the gap adjusting means to adjust the gap between the upper and lower plates.
 11. The apparatus as claimed in claim 10, wherein the gap adjusting means further comprises a stopper for restricting an upward motion of the upper plate support to allow the cell mixture layer to be maintained.
 12. A cell separation method, comprising the steps of: (a) creating a cell mixture containing specific cells tagged with magnetic beads into a cell mixture layer by adjusting a gap between upper and lower plates to be decreased such that the cell mixture which is accommodated in a cell mixture holding portion of the lower plate in an upwardly convex shape can be adsorbed in a bottom surface of the upper plate positioned opposite to the cell mixture holding portion of the lower plate; (b) moving the specific cells toward the upper plate by applying a magnetic field to the cell mixture layer created in step (a) from the upper plate and simultaneously moving cells other than the specific cells toward the lower plate by means of gravity; and (c) allowing the specific cells moved toward the upper plate and the other cells moved toward the lower plate in step (b) to be divided and then positioned in the bottom surface of the upper plate and the cell mixture holding portion of the lower plate, respectively, when the cell mixture layer is separated by increasing the gap between the upper and lower plates.
 13. The method as claimed in claim 12, further comprising the step of, after step (a), adjusting the gap between the upper and lower plates to maintain a thickness of the cell mixture layer at an optimal cell separation state.
 14. The method as claimed in claim 12, further comprising the steps of: (d1) creating a specific cell mixture layer by decreasing the gap between the upper and lower plates after removing the other cells divided and positioned in the lower plate in step (c) or replacing the lower plate with a new one and then injecting a buffer solution containing no cells in the lower plate; (e1) homogenizing the specific cell mixture layer by changing the gap between the upper and lower plates repeatedly several time while maintaining the specific cell mixture layer created in step (d1) (f1) moving the specific cells toward the upper plate by the magnetic field applied to the specific cell mixture layer homogenized in step (e1) from the upper plate and simultaneously moving the other cells in the specific cell mixture layer toward the lower plate by means of gravity; and (g1) allowing the specific cells moved toward the upper plate and the other cells moved toward the lower plate in step (f1) to be divided and then positioned in the bottom surface of the upper plate and the cell mixture holding portion of the lower plate, respectively, when the specific cell mixture layer is separated by increasing the gap between the upper and lower plates.
 15. The method as claimed in claim 12, further comprising the steps of: (d2) creating an other cell mixture layer by decreasing the gap between the upper and lower plates after removing the specific cells divided and positioned in the upper plate in step (c) or replacing the upper plate with a new one and then additionally injecting a buffer solution containing no cells in the lower plate; (e2) homogenizing the other cell mixture layer by changing the gap between the upper and lower plates repeatedly several times while maintaining the other cell mixture layer created in step (d2) (f2) moving the specific cells toward the upper plate by the magnetic field applied to the other cell mixture layer homogenized in step (e2) from the upper plate and simultaneously moving the other cells in the other cell mixture layer toward the lower plate by means of gravity; and (g2) allowing the specific cells moved toward the upper plate and the other cells moved toward the lower plate in step (f2) to be divided and then positioned in the bottom surface of the upper plate and the cell mixture holding portion of the lower plate, respectively, when the other cell mixture layer is separated by increasing the gap between the upper and lower plates. 